Members of the transforming growth factor-beta (TGF-beta) family of peptide growth factors, which include TGF-beta, bone morphogenetic proteins (BMPs) and activins, regulate a broad range of cellular processes from cell growth and differentiation to apoptosis. The signaling responses to TGF-beta and other family members are mediated by a heteromeric complex of two types of transmembrane serine/threonine kinase receptors at the cell surface, and their intracellular substrates, the Smad proteins. Proper TGF-beta superfamily signaling requires precise control of Smad functions. One of the important mechanisms that control Smad activity is ubiquitin-proteasome-mediated degradation. Previously, we and others identified Smurf1 and Smurf2, two new members of the HECT family of E3 ubiquitin ligases, as interacting partners for Smads, but the in vivo function of Smurfs and the selectivity of Smurfs towards Smad in specific signaling pathways remain to be determined. We continued our research interest in this area and began to address these important biological issues. We examined the role of Smurf1 in myogenic and osteogenic differentiation by taking advantage of the in vitro differentiation process of mouse C2C12 myoblast cells, which is subject to control by both TGF-beta and bone morphogenetic protein (BMP). We found that increased expression of Smurf1 promotes myogenic differentiation of C2C12 cells and blocks the BMP-induced osteogenic conversion but has no effect on the TGF-beta-induced differentiation arrest. Consistent with an inhibitory role in the BMP signaling pathway, the elevated Smurf1 markedly reduces the level of endogenous Smad5 while it leaves unaltered the levels of Smad2, Smad3 and Smad7, which are components of the TGF-beta pathway. Adding back Smad5 from a different source to the Smurf1-overexpressing cells restores the BMP-mediated osteoblast conversion. Finally, by depletion of endogenous Smurf1 through small interfering RNA-mediated RNA interference, we demonstrated that Smurf1 is required for the myogenic differentiation of C2C12 cells and plays an important regulatory role in the BMP-2-mediated osteoblast conversion. Although Smads are involved in most actions of the TGF-beta superfamily, many reports have suggested that TGF-beta may signal through alternative pathways. In order to characterize the mechanism of TGF-beta signaling through Smad-independent pathways and to understand the function of Smad-independent TGF-beta receptor signaling, we have generated a mutant TGF-beta type I receptor that is unable to activate Smads but retains kinase activity. We found that this mutant TGF-beta type I receptor is able to activate p38 kinase, and the p38 activation is required for TGF-beta induced apoptosis and epithelial to mesenchymal transition. These results indicate that the TGF-beta receptor exerts its signals through multiple intracellular pathways and provide first hand biochemical evidence to support the existence of Smad-independent TGF-beta receptor signaling. Currently we are working to identify downstream mediators that are responsible for Smad-independent TGF-beta receptor signaling. These studies could uncover novel molecular mechanisms that account for a number of Smad-independent TGF-beta signaling responses. In addition, we are also interested in how TGF-beta signaling converges with other pathways in response to growth factors and consequent activation of mitogen-activated protein kinase(MAPK) pathways. We would like to understand the role of this cross-talk in controlling TGF-beta-regulated gene transcription, cell proliferation, extracellular matrix production,apoptosis and tumor progression. In the meantime, a long term research program using mouse genetics to address the physiological and pathological roles of TGF-beta/Smad signaling in tumorigenesis has also been